Identification of tonoplast membrane proteins involved in cellular response to environmental stress by quantitative/comparative- and phospho-proteomics

蛋白质组分析是识别空泡蛋白和揭示它们在应激反应中的相关性的强大工具。在过去的研究资助期间，我们主要致力于建立一个分离高纯度空泡的平台，适用于基于质谱学的蛋白质组学。我们从低温培养的植物和对照植物中分离液泡，以研究冷驯化过程中液泡膜蛋白质组的变化。因此，液泡膜蛋白丰度的几个变化和蛋白磷酸化的变化被识别出来，为液泡在冷驯化中的生理相关性提供了第一个洞察力。通过这些蛋白质组分析，我们鉴定了几个存在于液泡膜中的未知蛋白质，并对其中一个进行了详细的鉴定。液泡膜铜转运蛋白COPT5属于真核细胞铜转运蛋白家族，催化铜离子从液泡中输出，并负责铜在拟南芥中的器官间分配。在拟议的第二个资助期，我们将扩大对干旱、盐、高温和渗透胁迫等其他非生物因素引起的液泡膜蛋白质组变化的比较分析。同时，我们将研究这些应激因素如何改变液泡膜蛋白的磷酸化，以了解这种翻译后蛋白修饰与液泡膜运输机制调控的相关性。最后，我们将研究单个液泡膜蛋白与植物胁迫反应的相关性，并努力阐明迄今未知的液泡膜蛋白的生化和生理功能。

Proteom analyses represent a formidable tool to identify vacuolar proteins and to enlighten their relevance in stress response. During the past period of the research grant we have mainly focused on establishing a platform to isolate highly pure vacuoles applicable for mass spectrometry based proteomics. We isolated vacuoles from cold incubated and control plants to investigate changes in the tonoplast proteome during the process of cold acclimation. Thereby several changes in the abundance of tonoplast proteins and alterations in protein phosphorylation were identified providing first insights into the physiological relevance of the vacuole on cold acclimation. By these proteome analyses we identified several unknown proteins residing in the tonoplast and characterized one of these in detail. The tonoplast copper transporter COPT5 belongs to the eukaryotic Ctr family, catalyses the export of copper ions from the vacuole and is responsible for the inter organ allocation of copper in Arabidopsis. In the proposed second funding period we will expand the comparative analysis of changes in the proteome of the tonoplast induced by other abiotic factors like drought, salt, heat and osmotic stress. In parallel we will investigate how such stress factors alter the phosphorylation of tonoplast proteins to understand the relevance of this post-translational protein modification on the regulation of tonoplast transport mechanisms. Finally, we will investigate the relevance of individual tonoplast proteins for the plants stress response and we will effort to clarify the biochemical and physiological function of so far unknown tonoplast proteins.